Sample Collection, Handling, and Logistics Manual

This manual is applicable for collection, handling, and logistics of formalin-fixed paraffin-embedded tissue samples for tests provided at HistoGeneX.

The procedures described here are considered as advisable and most optimal.

2. Responsibilities

It is the responsibility of the section head of the P&S unit to keep this procedure up to date and to inform stakeholders on changes made to this procedure.

3. Abbreviations, Definitions

FFPE

Formalin fixed Paraffin Embedded

g

Gram

l

Liters

ml

Milliliters

NBF

Neutral Buffered Formalin

P&S

Processing and Sectioning

4. Procedure

4.1. Sample collection

4.1.1 Neutral buffered formaldehyde 4%

Before initiation of sample collection, fixative and fixation containers should be at hand.

The preferred fixative is neutral buffered formalin which can be purchased or prepared in house.

For preparation of 4% neutral buffered formaldehyde for histology, perform the following process:

Formaldehyde (40% w/v)100 mL

Sodiumdihydrogenphosphate*H2O 4,0 g

Disodiumhydrogenphosphate 6,5 g

Distilled water fill up until 1L solution

Check pH, this has to be 7,4 ± 2,0 otherwise the solution is discarded.

4.1.2 Sampling (endoscopy/surgery room)

Different types of specimens can be collected for preparation of FFPE tissue samples.

The general rule accepted for optimal fixation is to prepare a minimum fixative volume of 10 times the biopsy/specimen volume and to use appropriate container sizes.

At HistoGeneX biopsies/specimens are classified into three classes (resection, punch biopsy, or needle core biopsy) which defines the processing conditions to paraffin (refer to 4.2).

The time to fixation is preferably shorter than 5 minutes.

The total fixation time should be more than 6 hours and should not exceed 48 hours (depending on the size, small to large specimens).

Complete sample collection, processing, storage and shipping log if applicable. A long-sleeved lab coat, gloves and safety goggles should be worn when handling human tissue or laboratory specimens for both biosafety precautions and prevention of contamination of the specimens.

4.1.3 Disposal of materials used in sample collection

Disposal of materials used in samples collection should be performed according to Good Laboratory Practices.

4.2. Sample handling - Tissue processing

Further fixation of samples may be necessary as this depends upon the size of the specimen.

When the maximally advised fixation time is reached, the sample is then dehydrated preferably in a series of alcohols and xylol, followed by infiltration of melted paraffin at no more than 60°C (± 2°C) as outlined in one of the schemes below:

Accepted methods for processing tissue are:

Step

Reagent

Temperature

Vacuum

Time

1*

Formalin

RT

3-45 h

2

Formalin

39±1°C

Yes

3 h

3

70% Alcohol

39±1°C

yes

1h

4

80% Alcohol

39±1°C

yes

1h

5

90% Alcohol

39±1°C

yes

1h

6

96% Alcohol

39±1°C

yes

1h

7

100% Alcohol

39±1°C

yes

1h

8

100% Alcohol

39±1°C

yes

1h

9

Xylene

39±1°C

yes

1:30h

10

Xylene

39±1°C

yes

1:30h

11

Paraffin

59±1°C

yes

0:50h

12

Paraffin

59±1°C

yes

1:05h

*Be aware: total fixation time should be 6-48 hours, including the fixation time in the automatic device.

OR

Step

Reagent

Temperature

Vacuum

Time

1*

Formalin

RT

6-48 h

2

Isopropanol

39±1°C

Yes

1:30 h

3

Isopropanol

39±1°C

yes

1h

4

Isopropanol

39±1°C

yes

1h

5

Isopropanol

39±1°C

yes

1h

6

Isopropanol

39±1°C

yes

1h

7

Isopropanol

39±1°C

yes

1:30h

8

Isopropanol

39±1°C

yes

1:30h

9

Toluol

39±1°C

yes

1h

10

Toluol

39±1°C

yes

1:30h

11

Paraffin

59±1°C

yes

1h

12

Paraffin

59±1°C

yes

1:30h

13

Paraffin

59±1°C

yes

1h

Preferably the block is handled according to preferred fixation method and timelines mentioned above, however, deviations will be accepted. In this case, a comment should be mentioned on the Oncology Request Form.

3. Preferably the block is handled according to the fixation method and timelines mentioned above, however, deviations will be accepted. In this case, a comment should be mentioned on the Oncology Request Form.

4.3.1.2 Slides of FFPE tissue

1. In case a paraffin embedded tissue block cannot be submitted, unstained and freshly cut slides from a FFPE tissue block, using a microtome, must be submitted (number of slides as specified per test).

If the slides are used for molecular analysis DNAse free sectioning is required.

All materials used for sectioning are cleaned with UltraClean Labcleaner. This includes the microtome (i.e. knife holder and handwheel), work surfaces, waterbath, petridish, spatula, lancet, labels and slide boxes. MilliPore water is replaced by Braun Water. A long-sleeved lab coat, gloves should be worn during sectioning.

2. Preferably, sectioning is performed as described above, however, deviations will be accepted. In this case, a comment should be mentioned on the Oncology Request Form.

3. Preferably use the SUPERFROST PLUS or ULTRA PLUS coated glass slides. If not available, other glass slides may be used, but make sure that these slides have been treated in order to provide a positively charged glass surface as this is necessary for proper adhesion of the tissue section. Pre-label the frosted area with unique identification code.

When small specimens (needle-core and punch) are to be collected on the glass slides or analytical testing involves fluorescent immunostaining, SUPERFROST PLUS GOLD slides are advised.

4. Float the sections on a water bath at 45°C-50°C ±2°C keeping track of the order in which the sections were cut. The water bath should contain distilled water without gelatin or other protein-rich products, as the latter damage the adhesive coating of the coated glass slides.

5. Mount each section on the coated side of an appropriately labelled slide ensuring that the patient ID and the patient ID on the label match. Mount the section at the opposite end to the frosted area, about 1/3 from the edge. For larger specimens mount the sample from this point towards the frosted area. For small specimens, a series of sections may be mounted, starting from this same point. Always collect consecutive sections on consecutively labelled microscope slides.

6. Place the glass slide with mounted tissue section between two pre-wetted filter papers (Attention: sections can stick to the filter paper when too wet/dry).

7. Allow sections to attach to the glass slide by gently pressing and rolling your fist over the filter paper.

8. Place microscope slides in an appropriately labeled slide box.

9. Place slide racks/boxes in the oven at 60°C ± 2°C with ventilation (no humidification) during 30 ± 5 minutes for DNA/RNA-applications and 1-2 hours for other applications as requested in the appropriate procedures.

10. Store and ship samples at ambient temperature.

4.3.2. Completion of Oncology Request Form

Submission of samples for diagnostic testing must always be accompanied with the Oncology Request Form. Guidelines for completion of this form can be found in Instructions for completion of Oncology Request Form. As stated in these guidelines, primary samples lacking proper identification shall not be accepted or processed by the laboratory. In addition, verbal request for sample examination will not be accepted. Only tests requested in the Oncology Request form will be performed. Additional tests can only be performed if an additional Oncology Request Form is submitted. Retesting of samples can be done without submission of a new Oncology Request form only if the initial test failed due to analytical problems.

4.3.3. Logistics

Tissue blocks and slides can be shipped to HistoGeneX by using regular mail or a courier and must be securely packaged, for example in a protective envelope. Both tissue blocks and slides can be shipped at room temperature. The shipped samples must always be accompanied with the correctly completed Oncology Request Form.

5. Sample reception

Samples can be received from Monday to Friday between 08.00h and 15.00h.

For those samples that arrive after 15.00h, calculation of the Turn-Around-Time (TAT) will start the day after delivery.

Samples that do not meet the acceptance criteria set in the Instructions for completion of Oncology Request Form will not be accepted for processing and testing in the laboratory. However, if compromised samples are accepted, this will be clearly mentioned in the sample reception log and the final report. Furthermore, caution should be taken while interpreting the results.

Samples will be processed according to fixed turnaround times as specified per test. No exceptions will be made if samples are marked as urgent.

6. Storage of samples

FFPE tissue blocks will be sent back to the requesting laboratory upon finalization of the requested test(s). Remaining tissue sections, however, will not be sent back and will be stored at 2-8 °C unless otherwise requested.